Three separation techniques have been employed to demonstrate that biliary lipids in human gallbladder bile are found in association with protein-lipid complexes. The initial isolation indicating the existence of this entity was accomplished by an Ultracentrifuge (UC) experiment. Bile was top-layered on a 12% solution of a sucrose polymer, Ficoll (MW 400,000) (d equals 1.040) in UC tubes. During the UC run, a protein:lipid-rich bilirubin stained band migrated into a spontaneously-formed continuous density gradient. The position of this band could be shifted upward or downward depending upon the total g-exposure thus also shifting the gradient slope in the UC tube. But the position of the band in the gradient (d equals 1.015) was unchanged. Most of the protein detectable in native bile was found in the lipid-rich band. Other isolation techniques supporting the presence of protein-lipid interactions included the use of Sepharose CL-6B column chromatography of whole untreated human gallbladder bile and similar chromatography of the density gradient isolated-protein:lipid rich band in which the original bile salt concentration has been exogenously restored. The isolated UC band fractions and protein:lipid-rich column fractions following lyophilization and delipidation were then studied by analytical slab SDS-PAGE. These results indicated that virtually all of the protein species detectable in whole native bile are involved in protein-lipid interactions. This is the clearest demonstration to date of the existence and chemical composition of these interacting complexes, but their biologic significance remains to be established. In the present proposal, we plan to further probe these isolated complexes to determine among other issues whether specific protein species have higher interaction potential than most of the others, whether "bile specific" proteins are involved, whether reconstitution is possible thereby to learn more about biologic function and lastly whether a qualitative or quantitative difference in the protein component may play a role in the pathogenesis of cholesterol cholelithiasis.